|Publication number||US7767446 B2|
|Application number||US 11/227,489|
|Publication date||Aug 3, 2010|
|Filing date||Sep 16, 2005|
|Priority date||Sep 16, 2004|
|Also published as||US20060110822|
|Publication number||11227489, 227489, US 7767446 B2, US 7767446B2, US-B2-7767446, US7767446 B2, US7767446B2|
|Inventors||Neil F. Robbins, Jon Rowley, Mark Quinto, Abel Z. Hastings, Bryan G. Towns, Bradley R. Snodgrass|
|Original Assignee||Becton, Dickinson And Company|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (14), Non-Patent Citations (1), Referenced by (11), Classifications (11), Legal Events (4)|
|External Links: USPTO, USPTO Assignment, Espacenet|
The present application claims benefit to U.S. Provisional Patent Application Ser. No. 60/714,516, filed Sep. 16, 2004, and U.S. Provisional Patent Application Ser. No. 60/699,849, filed Jul. 18, 2005, the entire contents of which are incorporated herein.
The present invention relates generally to the field of bioreactors, and, more particularly, to a system and method for culturing cells under perfusion flow, in a single chamber or in a high throughput format.
Recent developments in cell/tissue engineering have recognized benefits to growing and studying cells in dynamic environments. Spinner flasks, rotary devices, perfusion bioreactors, or fluid sheer chambers have all been used to enhance nutrient and metabolite diffusion to and from cells. The mechanical aspects of fluid sheer forces have also been shown to trigger second messenger signals and alter cellular gene expression. While these new culture conditions have been recognized to affect cell functions (growth, signaling, morphology, differentiation, etc.), devices for studying these environments have not been translated to high throughput platforms. Furthermore, systems that incorporate three-dimensional scaffolds with highly aligned pores for long-range control over fluid flow paths have also not been established.
Fluid flow was first established as a regulator of cellular gene expression in two-dimensional culture systems with flowing culture medium over cells adherent to glass slides. Cells respond to the fluid sheer by aligning in the direction of the force, and altering their gene expression. These two-dimensional devices are now commercially available from, for example, Flex-Cell International, as well as other vendors. Fluid flow studies have recently been translated to three-dimensional scaffolds, and it has been established that fluid sheer is another important factor in maintaining hepatocyte and bone differentiation. The true importance of fluid flow as an environmental signaling factor, however, has not been fully appreciated because it is difficult to screen against or study in conjunction with a plethora of other environmental cues that are known to alter cell function including but not limited to signaling factors such as growth factors, ECMs, cytokines, media factors, and small molecules to name a few. For example, to date, all of the devices designed to study how these forces affect cell cultures are one-pot or single chamber devices. These devices may be utilized to study how rotation or fluid sheer forces affect cells under one condition at a time, but not under different or varying conditions, which greatly limits the utility of these devices. Accordingly, current devices are not suitable for performing medium or high throughput experimentation for optimization of conditions for controllable cell phenotype, or for testing substances such as molecules of unknown function for altering specific functions in highly relevant cell or engineered tissue cultures.
Furthermore, in the field of drug discovery, the use of primary human cells to study ADMETox (ADMETox is an acronym for set of analyses that measure the absorption, distribution, metabolism, elimination and toxicity of a drug candidate) properties of drugs is highly desirable. This is due to the fact that whole animal studies are expensive, and results are not always predictive of responses in man. In vitro study of primary human cells is attractive due to the economics of the approach, and the fact that data from human cells should be more relevant than animal data. Unfortunately, the culture of primary human cells is extremely difficult for most cell types, and there are few model systems that are capable of creating relevant models of in vivo tissues and organs. As an intermediate between whole animals and primary cells, tissue or organ slices offer an alternative that keeps cells in their native setting (not dissociating them from their microenvironment), while allowing for in vitro testing of xenobiotic effects on cell viability, metabolism, and other ADMET-type aspects that one desires. For example, liver slices are often utilized for measuring liver-specific drug toxicity, as well as CYP induction.
In vitro culture of tissue slices also has several challenges. For example, one significant challenge is the high metabolic rates and nutrient requirements that tissue slices need in vitro. Since the tissue slices require a large nutrient load, it is necessary to culture these slices in large quantities of medium. However, the more medium that one adds to a culture increases the diffusion distance of oxygen to the extent that the rate of consumption by the tissue is greater than the diffusion of oxygen, leading to hypoxic conditions and cell death. There is, therefore, a great need for bioreactor-type devices that enhance nutrient and metabolite transport while maintaining a medium-to-high throughput parallel testing format.
A need exists for a system and method for culturing cells under fluid perfusion in medium to high throughput format, to test and/or discover how new environments alter the ability of cells to respond to other chemical or physical cues in the presence of fluid sheer and to facilitate the systematic and high throughput discovery of dynamic cell culture conditions for cell growth and differentiation, and then utilizing these optimized environments for creating in vitro engineered tissues for therapeutic, diagnostic, or research purposes. A need also exists for a perfusion system that allows for the dynamic and multiplexed culture of a variety of tissue or organ slices for ADMET and tissue culture applications.
The present invention is directed to a bioreactor system including a perfusion unit, a pumping unit in fluid communication with the perfusion unit, and a fluid source unit in fluid communication with the pumping unit. The perfusion unit includes an array of cell wells configured to contain cell cultures and the fluid source unit includes an array of media wells configured to contain cell culture media. The pumping unit includes an array of pumping elements in fluid communication with the cell wells and media wells and is configured to pump cell culture media from the media cells to the cell wells.
In a preferred embodiment, each of said cell wells is adapted and configured to contain a scaffold having a porous structure. In one embodiment, the scaffold is a two-dimensional scaffold. In another embodiment, the scaffold is a three-dimensional scaffold. In one embodiment, the three-dimensional scaffold may include directionally aligned pores.
In one embodiment, the fluid is deliverable directly into the internal structure of said scaffold. In another embodiment, a return pathway is provided for the fluid to flow from the array of cell wells to the array of media wells. In a preferred embodiment, each pathway is in fluid communication with a single cell well and a single media well.
In another embodiment, the perfusion unit is removably couplable to the pumping unit and each pumping element may comprise a fluid stem having a fluid port therein. Each stem may be configured to extend into the cell wells. In another aspect of the invention, each cell well may include a scaffold coupled thereto configured to receive a portion of the stem internal thereto. The fluid source unit is also removably couplable to the pumping unit.
The present invention is also directed to a method of growing cells, comprising pumping cell culture media from a first array of wells of a fluid source unit into a second array of wells of a perfusion unit, wherein each well of the perfusion unit is adapted and configured to house a cell adherent structure. In one embodiment the method further comprises the step of perfusing the media into and through a scaffold. In one embodiment, the cell adherent structure comprises a two-dimensional scaffold, and in another embodiment the cell adherent structure comprises a three-dimensional scaffold. The first array of wells is in fluid communication with the second array of wells for the return of media to the second array of wells. In another method, each well of the first array of wells is in singular fluid communication with a corresponding well of the second array of wells.
The present invention is also directed to a perfusion bioreactor system, including an array of bioreactor units. Each bioreactor unit includes a cell adherent structure in fluid communication with a fluid stored in a fluid reservoir and, in operation, fluid flows from the fluid source directly into and through the cell adherent structure. In one variation, the cell adherent structure is a three-dimensional scaffold having a porous structure, and in another variation the cell adherent structure is a two-dimensional scaffold. In another preferred embodiment, the cell adherent structure is fluidly interconnected to the fluid reservoir by a pumping unit.
In the drawings:
The present invention relates to bioreactors generally, and, more particularly, to a system and method for culturing cell specimens under perfusion flow, in a single chamber or in a high throughput format for the high throughput discovery of complex environments for controlling cell function and engineered tissue development. The present invention may also be utilized for creating highly relevant cell cultures and systems for direct drug testing on cells in dynamic cell cultures, for drug discovery, drug testing, or ADMETox applications.
In a preferred embodiment, perfusion unit 12 is a multi-well plate including a plurality of main chambers or wells 18 configured to house or contain a cell culture. Similarly, the fluid source unit 14 may comprise one or more separate multi-well plates including a plurality of fluid reservoir chambers or wells 20 to store fluid, such as cell culture media. In operation, each main chamber or well 18 is in fluid communication with a corresponding individual fluid reservoir chamber 20. In a preferred embodiment, top perfusion unit 12 and fluid source unit 14 include 24 chambers or wells, however, in alternative embodiments any number of chambers or wells may be provided. For example, the wells of top perfusion unit 12 and the fluid source unit 18 may be miniaturized to comprise 48 wells per plate, 96 wells per plate, or smaller. Similarly, pumping components may be miniaturized to comprise a smaller bioreactor system with a similar footprint, or increasing the footprint to have more individual perfusion units on one system. Referring to
In another preferred embodiment, perfusion unit 12 and fluid source unit 14 are preferably configured and dimensioned to be removably coupled to pumping unit 16. Accordingly, perfusion unit 12 and fluid source unit 14 may be interchangeable components of the system, such that a plurality of like units or plates may be exchanged or removably coupled to pumping unit 16 as desired. For example, the fluid source unit 14 is configured to be removably coupled to the pumping unit 16 such that the fluid source unit 14 may be re-usable or disposable for media addition. Similarly, perfusion unit 12 may be removed from one pumping unit 16 to another to associate cell cultures with different fluid/dynamic environments.
Pumping unit 16 comprises an array of fluid connectors and/or hardware components to fluidly connect each main chamber 18 with each fluid reservoir chamber 20. In a preferred embodiment, pumping unit or station 16 may comprise any hardware components suitable for transferring or pumping fluid from the fluid source unit 14 to the perfusion unit 12 such as, for example, motorized pump(s), valves, tubes, pipes, or other devices or means for pumping or transferring the fluid. Generally, any type of pumping mechanism may be used, including but not limited to peristaltic, centrifugal, vibrating, piezo, or an air or fluid driven pumping mechanism, or individual electronic pumps wherein each perfusion unit could be programmed with a different pumping rate. In a particular preferred embodiment, shown in
In a preferred embodiment, the cell adherent structure is coupled to the main chamber about a fluid port 44 such that the fluid flows directly into or about the cell adherent structure. For example, a three-dimensional scaffold 22 may be coupled, molded, bonded, synthesized, or otherwise attached to the main chamber 18 such that a stem or fluid port 44 extends into the central portion or interior of the scaffold when, for example, perfusion plate 12 is coupled to pumping unit 16. In another preferred embodiment, each main chamber 18 of perfusion plate 12 is configured to receive scaffolds that may be coupled, fastened, or otherwise connected to a portion of each main chamber 18 by any suitable means known to those skilled in the art. In one preferred embodiment, scaffold 22 may be releasably plugged into or attached to main chamber 18.
The scaffolds can be made from any type of polymer, ceramic, metal or mixture of any type suitable for adhering cells thereto. In a preferred embodiment, the scaffold is made from a hydrogel-based material, which may be synthesized from covalently crosslinked alginate, hyalrunic acid or a blend of the two polysaccharides at any mixing percentage as desired. For example, the mixing percentage may be tailored to achieve a desired degradation profile for the final application. In alternate embodiments, the scaffolds may be made of other suitable materials, such as those disclosed in U.S. Patent Publication No. 2004/0147016 entitled “Programmable scaffold and methods for making and using same”, the entire contents of which are incorporated by reference. In one preferred embodiment, the scaffold may be a porous structure having randomly aligned pores. In alternative embodiments, scaffolds may be used that have directionally aligned pores such that a less random pore pattern may be attained and fluid flow may be further assured of navigating or flowing through all of the pores of the scaffold. In alternate embodiments, the scaffolds may be modified with any number or type of cell signaling or cell interacting molecule, such as those disclosed in U.S. Patent Publication No. 2004/0147016, entitled “Programmable scaffold and methods for making and using same,” the entire contents of which are incorporated by reference.
In operation, fluid is pumped directly into the internal scaffold structure and may perfuse or flow from the interior 46 of scaffold 22 to the exterior 48 of scaffold 22. In one preferred embodiment, fluid is pumped at a rate ranging from about 10 to 0.1 milliliters per minute. In this regard, fluid may readily flow through the internal pores of the scaffold as opposed to circumventing the scaffold or flowing mainly along the exterior of the scaffold. The enhanced diffusion mass transport provided by the perfused fluid flow advantageously allows metabolites and nutrients to diffuse into and out of scaffold 22. In this regard, perfusion culture permits long term tissue engineering experiments allowing growth of high density cell cultures to mimic tissues.
In prior art devices where fluid is permitted to circumvent the scaffold, severe oxygen limitations may be caused because oxygen is consumed by the cells adhered upon the outside of the scaffold and cells adhered upon the inside of the scaffold may be oxygen starved.
In one embodiment, cell specimen 70 has a cylindrical or disc shape and may be held in place in main chamber 18, for example, between a pair of washers 72. Washers 72 include a central opening to permit fluid flow therethrough. In operation, fluid may flow through port 44 and perfuse through cell specimen 70 and exit through the central opening of the top washer 72 and return via return pathway 75. In this regard, the present embodiment is configured to keep slices or cell specimens emerged at all times in media, while exposing the tissue or cell specimen to fluid flow similar to in vivo conditions and enhancing gas and nutrient transfer. In a preferred embodiment, the present system facilitates the maintaining of cell viability, and the maintaining of the specimens or tissue slices in a format for drug testing. The configuration of this embodiment may be advantageously utilized with, for example, tissue slices or scaffolds made of polymer or ceramic material or other materials that cannot be synthesized in place.
The pumping device 84 of the present embodiment generally comprises a pressure chamber 96 having an air inlet 98 and a flexible diaphragm 100 that interfaces with the bottom reservoir plate 82. As best seen in
The pumping device 114 of the present embodiment generally comprises a pressure chamber 126 having an air inlet 128 and a plurality of flexible diaphragms 130 that surround flexible passages 132. Flexible passages 132 extend between the fluid reservoirs 122 of the bottom plate and a pair of one-way valves or check valves 125 aligned with the inlet and outlet portions 118, 120 of the perfusion plate 116. As best seen in
With respect to all of the aforementioned multi-well bioreactor systems, different cell types may be cultured in the same set of wells. For example, in the embodiment of
Utilizing the aforementioned multi-well bioreactor systems of the present invention, unique experiments may be studied that incorporate fluid flow. For example, multiple parallel experiments may be performed having substantially similar fluid flow characteristics. In this regard, highly complex environments may be created to perform experiments in a medium throughput or high throughput format. One skilled in the art could also create cultures consisting of several types of cell and tissue systems in fluid communication for studying complex metabolic diseases such as diabetes, obesity, and cardiovascular diseases to name a few. In one particular application for optimizing cell signaling environments, a variety of soluble and non-soluble signaling molecules consisting of growth factors, cytokines, extracellular matrix molecules, etc., may be tested at different concentrations, different mixing ratios, and at various times to facilitate the discovery of an optimal combination of factors to obtain a fully differentiated cell culture in vitro. These, environments may be created utilizing a variety of parenchymal cells and non-parenchymal cells from tissues including bone marrow, vasculature, skin, pancreas, liver, bone, cartilage, smooth muscle, cardiac muscle, skeletal muscle, kidney, etc. In another embodiment, cells such as endothelial cells may be used to create vascularization with the host. In alternate embodiments, one skilled in the art could also create cultures consisting of several types of tissue systems for studying complex metabolic diseases such as the metabolic syndrome. In alternative applications, several cell types may be incorporated to study fluid sheer and perfusion, for example, to determine fluid flow that most likely promotes cell-type segregation for vasculorgenesis and tissue development. In another application, the cells or tissue grown in the multi-well design may be used as a platform for testing drugs in a medium to high throughput format for direct drug testing on cells in dynamic cell cultures, either for drug discovery, drug testing, or ADMETox applications. Furthermore, sensing technology may be incorporated into the bioreactor system. For example, biosensing technology for sensing important cell culture variables such as glucose, ammonia, urea, pH, or general fluorescent detectors for monitoring metabolism of fluorescent compounds may be utilized with the system.
In one exemplary variation or application, a perfusion unit 12 may be used to grow cell cultures with preset conditions or particularly desirable characteristics which can then be later used for further experimentation and or discovery. The modularity and interchangeability of perfusion unit 12 advantageously permits the shipment and or transfer of a plurality of cell cultures which can be easily remounted on another pumping station 16 or similar device to perform further experimentation and/or drug testing or discovery.
Referring now to
Each well unit 210 generally comprises a frustoconical or tapered body 230 exetending distally from the top of carrier 205 and includes a scaffold holding chamber 232 at the distal end 234. A cell adherent structure or scaffold 220 is preferably housed or held within each well unit 210 to facilitate high density cell culture growth. In a preferred embodiment, the cell adherent structure is coupled or loaded into to the well unit 210 about a distal end 234. For example, a three-dimensional scaffold 220 may be coupled, molded, bonded, synthesized, or otherwise attached to the distal chamber 232. In one preferred embodiment, scaffold 220 may be releasably plugged into or attached to chamber 232 for example by friction fit.
Scaffold handling system 201 and carrier 205 of
Sidewalls or flanges 216, 218 of carrier 205 extend distally from the lateral sides of carrier 205 and are configured and dimensioned to extend about the lateral outside of the multi-well plate to accurately mate carrier 205 with the 24-well plate. As best seen in
In yet another embodiment, scaffold handling system 201 and carrier 205 of
Referring again to
While the invention has been described in conjunction with specific embodiments and examples thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art upon reading the present disclosure. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
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|U.S. Classification||435/299.2, 435/293.1, 435/289.1|
|International Classification||C12M3/04, C12M1/14|
|Cooperative Classification||C12M23/12, C12M25/14, C12M29/10|
|European Classification||C12M23/12, C12M29/10, C12M25/14|
|Mar 22, 2006||AS||Assignment|
Owner name: BECTON, DICKINSON AND COMPANY, NEW JERSEY
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ROBBINS, NEIL F.;ROWLEY, JON;QUINTO, MARK;AND OTHERS;REEL/FRAME:017359/0836;SIGNING DATES FROM 20060124 TO 20060306
Owner name: BECTON, DICKINSON AND COMPANY, NEW JERSEY
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ROBBINS, NEIL F.;ROWLEY, JON;QUINTO, MARK;AND OTHERS;SIGNING DATES FROM 20060124 TO 20060306;REEL/FRAME:017359/0836
|Feb 10, 2014||AS||Assignment|
Owner name: ORGANOVO, INC., CALIFORNIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BECTON, DICKINSON AND COMPANY;REEL/FRAME:032187/0714
Effective date: 20130118
|Feb 26, 2014||FPAY||Fee payment|
Year of fee payment: 4
|Feb 26, 2014||SULP||Surcharge for late payment|